Quantum Dot-Conjugated SARS-CoV-2 Spike Pseudo-Virions Enable Tracking of Angiotensin Converting Enzyme 2 Binding and Endocytosis

Abstract

The first step of SARS-CoV-2 infection is binding of the spike protein’s receptor binding domain to the host cell’s ACE2 receptor on the plasma membrane. Here, we have generated a versatile imaging probe using recombinant Spike receptor binding domain conjugated to fluorescent quantum dots (QDs). This probe is capable of engaging in energy transfer quenching with ACE2-conjugated gold nanoparticles to enable monitoring of the binding event in solution. Neutralizing antibodies and recombinant human ACE2 blocked quenching, demonstrating a specific binding interaction. In cells transfected with ACE2-GFP, we observed immediate binding of the probe on the cell surface followed by endocytosis. Neutralizing antibodies and ACE2-Fc fully prevented binding and endocytosis with low nanomolar potency. Importantly, we will be able to use this QD nanoparticle probe to identify and validate inhibitors of the SARS-CoV-2 Spike and ACE2 receptor binding in human cells. This work enables facile, rapid, and high-throughput cell-based screening of inhibitors for coronavirus Spike-mediated cell recognition and entry.

The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules

“…We previously developed a quantum dot-conjugated SARS-CoV-2 receptor binding domain nanoparticle (QD-RBD) that tracks viral spike binding to ACE2 and subsequent endocytosis into the cell. 34 Upon treating cells with QD-RBD, the nanoparticle rapidly binds ACE2 on the cell surface and induces endocytosis. In ACE2-GFP expressing cells, both the QD-RBD and ACE2 can be visualized using fluorescence microscopy.…”

NeutrobodyPlex - Nanobodies to monitor a SARS-CoV-2 neutralizing immune response

“…Additionally, we included a non-specific Nb (GFP-Nb) as negative control and two inhibiting mouse antibodies 24 as positive controls. Data obtained through this multiplex binding assay showed that 8 of the 10 analyzed Nbs inhibit ACE2 binding to all tested SARS-CoV-2 antigens ( Supplementary Figure 2).…”

Multifunctional angiotensin converting enzyme 2, the SARS-CoV-2 entry receptor, and critical appraisal of its role in acute lung injury

“…Binding of both SARS-CoV and SARS-CoV-2 to ACE2 leads to downregulation of ACE2 expression on the cell surface [ 112 , [165] , [166] , [167] , [168] ]. This is due to endocytosis of ACE2 bound to the virus and decreased ACE2 transcription in the host cell [ 149 , 150 , 166 , 169 , 170 ]. In addition, non-endocytotic internalization of the SARS-CoV-ACE2 complex, involving the cleavage of the ACE2 ectodomain by cell protease TMPRSS2 [ 152 , 171 ], causes further downregulation of ACE2 on the host cell surface.…”